Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation ...events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.
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•dCas9-Tet1 and -Dnmt3a enable precise editing of CpG methylation in vitro and in vivo•Targeted demethylation of BDNF promoter IV activates BDNF in neurons•Targeted enhancer demethylation facilitates MyoD-induced muscle cell reprogramming•Targeted de novo methylation of CTCF motifs alters CTCF-mediated gene loops
DNA methylation patterns can be specifically altered in mammalian cells using CRISPR/Cas9-based approaches.
Editing the Epigenome to Tackle Brain Disorders Liu, X. Shawn; Jaenisch, Rudolf
Trends in neurosciences (Regular ed.),
December 2019, 2019-12-00, 20191201, Letnik:
42, Številka:
12
Journal Article
Recenzirano
Genetic studies of epigenetic modifiers such as DNA methyltransferases and histone acetyltransferases have revealed a critical role for epigenetic regulation during brain development and function. ...Alteration of epigenetic modifications have been documented in a variety of brain disorders, including neurodevelopmental, psychiatric, and neurodegenerative diseases. Development of epigenome editing tools enables a functional dissection of the link between altered epigenetic changes and disease outcomes. Here, we review the development of epigenome editing tools, summarize proof of concept applications focusing on brain disease-associated genes, and discuss the promising application and challenges of epigenome editing to tackle brain disorders.
Current understanding of the epigenome is based on the systematic profiling of the epigenetic landscape in multiple tissues during development and pathogenesis of diseases.Development and application of epigenome editing tools has been accelerated by the discovery of the CRISPR/Cas9 system, allowing for locus-specific targeting of epigenetic effectors to any given genomic locus, through easy design and assembly of single guide RNA.Epigenome editing tools enable us to distinguish correlation and causality of epigenetic events associated with brain diseases.Off-target effects and stability of epigenome editing are two major considerations for any therapeutic application.Expansion of editing tools by introduction of other orthologs in the CRISPR/Cas systems will allow for simultaneous modifications of multiple genomic loci at different layers of epigenetic marks, enabling us to tackle polygenic disorders.
Microenvironmental oxygen (O(2)) regulates stem cell activity, and a hypoxic niche with low oxygen levels has been reported in multiple stem cell types. Satellite cells are muscle-resident stem cells ...that maintain the homeostasis and mediate the regeneration of skeletal muscles. We demonstrate here that hypoxic culture conditions favor the quiescence of satellite cell-derived primary myoblasts by upregulating Pax7, a key regulator of satellite cell self-renewal, and downregulating MyoD and myogenin. During myoblast division, hypoxia promotes asymmetric self-renewal divisions and inhibits asymmetric differentiation divisions without affecting the overall rate of proliferation. Mechanistic studies reveal that hypoxia activates the Notch signaling pathway, which subsequently represses the expression of miR-1 and miR-206 through canonical Hes/Hey proteins, leading to increased levels of Pax7. More importantly, hypoxia conditioning enhances the efficiency of myoblast transplantation and the self-renewal of implanted cells. Given the robust effects of hypoxia on maintaining the quiescence and promoting the self-renewal of cultured myoblasts, we predict that oxygen levels in the satellite cell niche play a central role in precisely balancing quiescence versus activation, and self-renewal versus differentiation, in muscle stem cells in vivo.
Pancreatic ductal adenocarcinoma (PDA) is the most lethal of common human malignancies, with no truly effective therapies for advanced disease. Preclinical studies have suggested a therapeutic ...benefit of targeting the Hedgehog (Hh) signaling pathway, which is activated throughout the course of PDA progression by expression of Hh ligands in the neoplastic epithelium and paracrine response in the stromal fibroblasts. Clinical trials to test this possibility, however, have yielded disappointing results. To further investigate the role of Hh signaling in the formation of PDA and its precursor lesion, pancreatic intraepithelial neoplasia (PanIN), we examined the effects of genetic or pharmacologic inhibition of Hh pathway activity in three distinct genetically engineered mouse models and found that Hh pathway inhibition accelerates rather than delays progression of oncogenic Kras-driven disease. Notably, pharmacologic inhibition of Hh pathway activity affected the balance between epithelial and stromal elements, suppressing stromal desmoplasia but also causing accelerated growth of the PanIN epithelium. In striking contrast, pathway activation using a small molecule agonist caused stromal hyperplasia and reduced epithelial proliferation. These results indicate that stromal response to Hh signaling is protective against PDA and that pharmacologic activation of pathway response can slow tumorigenesis. Our results provide evidence for a restraining role of stroma in PDA progression, suggesting an explanation for the failure of Hh inhibitors in clinical trials and pointing to the possibility of a novel type of therapeutic intervention.
Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in ...the 5′ UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.
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•Targeted demethylation of CGG repeats by dCas9-Tet1 reactivates FMR1 in FXS cells•Demethylation of CGG repeats induces an active chromatin status for FMR1 promoter•Methylation-edited FXS neurons behave similarly as wild-type neurons•FMR1 reactivation by dCas9-Tet1 is sustainable in a human/mouse chimeric model
Rescue of fragile X syndrome neurons by CRISPR-mediated DNA methylation editing of the FMR1 gene.
As a universal mechanical cue, shear stress plays essential roles in many physiological processes, ranging from vascular morphogenesis and remodeling to renal transport and airway barrier function. ...Disrupted shear stress is commonly regarded as a major contributor to various human diseases such as atherosclerosis, hypertension, and chronic kidney disease. Despite the importance of shear stress in physiology and pathophysiology, our current understanding of mechanosensors that sense shear stress is far from complete. An increasing number of candidate mechanosensors have been proposed to mediate shear stress sensing in distinct cell types, including G protein-coupled receptors (GPCRs), G proteins, receptor tyrosine kinases, ion channels, glycocalyx proteins, and junctional proteins. Although multiple types of mechanosensors might be able to convert shear stress into downstream biochemical signaling events, in this review, we will focus on discussing the mechanosensitive GPCRs (angiotensin II type 1 receptor, GPR68, histamine H1 receptor, adhesion GPCRs) and ion channels (Piezo, TRP) that have been reported to be directly activated by shear stress.
Secondary analysis of the 1998 Medical Expenditure Panel Survey.
To estimate total health care expenditures incurred by individuals with back pain in the United States, calculate the incremental ...expenditures attributable to back pain among these individuals, and describe health care expenditure patterns of individuals with back pain.
There is a lack of updated information on health care expenditures and expenditure patterns for individuals with back pain in the United States.
This study used data from the 1998 Medical Expenditure Panel Survey, a national survey on health care utilization and expenditures. Total health care expenditures and per-capita expenditures among individuals with back pain were calculated. Multivariate regression models were used to estimate the incremental expenditures attributable to back pain. The expenditure patterns were examined by stratifying individuals with back pain by sociodemographic characteristics and medical diagnosis, and calculating per-capita expenditures for each stratum.
In 1998, total health care expenditures incurred by individuals with back pain in the United States reached 90.7 billion dollars and total incremental expenditures attributable to back pain among these persons were approximately 26.3 billion dollars. On average, individuals with back pain incurred health care expenditures about 60% higher than individuals without back pain (3,498 dollars vs. 2,178 dollars). Among back pain individuals, at least 75% of service expenditures were attributed to those with top 25% expenditure, and per-capita expenditures were generally higher for those who were older, female, white, medically insured, or suffered from disc disorders.
Health care expenditures for back pain in the United States in 1998 were substantial. The expenditures demonstrated wide variations among individuals with different clinical, demographic, and socioeconomic characteristics.
Noxious pH triggers pungent taste and nocifensive behavior. While the mechanisms underlying acidic pH sensation have been extensively characterized, little is known about how animals sense alkaline ...pH in the environment. TMC genes encode a family of evolutionarily conserved membrane proteins whose functions are largely unknown. Here, we characterize C. elegans TMC-1, which was suggested to form a Na+-sensitive channel mediating salt chemosensation. Interestingly, we find that TMC-1 is required for worms to avoid noxious alkaline environment. Alkaline pH evokes an inward current in nociceptive neurons, which is primarily mediated by TMC-1 and to a lesser extent by the TRP channel OSM-9. However, unlike OSM-9, which is sensitive to both acidic and alkaline pH, TMC-1 is only required for alkali-activated current, revealing a specificity for alkaline sensation. Ectopic expression of TMC-1 confers alkaline sensitivity to alkali-insensitive cells. Our results identify an unexpected role for TMCs in alkaline sensation and nociception.
•Little is known about how animals sense alkaline pH in the environment•TMC-1 is required for C. elegans to avoid noxious alkaline environment•TMC-1 is required for alkali-activated currents in nociceptive neurons•Ectopic expression of TMC-1 confers alkaline sensitivity to alkali-insensitive cells
Little is known about how animals sense alkali in the environment. Wang et al. report that TMC-1, an evolutionarily conserved membrane protein, mediates alkaline sensation in C. elegans by functioning as an essential subunit of an alkali-activated channel.
DNA methylation is a key epigenetic mechanism underlying many biological processes, and its aberrant regulation has been tightly associated with various human diseases. Precise manipulation of DNA ...methylation holds the promise to advance our understanding of this critical mechanism and to develop novel therapeutic methods. Previously, we were only able to alter genome-wide DNA methylation by treating with small molecules (e.g., 5-Aza-2-deoxycytidine) or perturbing relevant genes (e.g., DNA methyltransferase) targetlessly, which makes it challenging to investigate the functional significance of this epigenetic mark at specific genomic loci. By fusing the catalytic domain of a key enzyme in the DNA demethylation process (Ten-eleven translocation dioxygenases 1, Tet1) with a reprogrammable sequence-specific DNA-targeting molecular protein, dCas9, we developed a DNA methylation editing tool (dCas9-Tet1) to demethylate specific genomic loci in a targeted manner. This dCas9-Tet1 system allows us to study the role of DNA methylation at almost any given loci with only the replacement of a single-guide RNA. Here, we describe a protocol that enables modular and scalable manipulation of DNA methylation at specific genomic loci in various cell cultures with high efficiency and specificity using the dCas9-Tet1 system. Key features • Precisely editing the DNA methylation of specific genomic loci in a targeted manner. • Fine-tuning gene expression without changing DNA sequence. • Applicable to many types of cell cultures and with the potential for ex vitro and in vivo applications.
Aging is characterized by a progressive decline in multiple physiological functions (i.e., functional aging). As animals age, they exhibit a gradual loss in motor activity, but the underlying ...mechanisms remain unclear. Here we approach this question in C. elegans by functionally characterizing its aging nervous system and muscles. We find that motor neurons exhibit a progressive functional decline, beginning in early life. Surprisingly, body-wall muscles, which were previously thought to undergo functional aging, do not manifest such a decline until mid-late life. Notably, motor neurons first develop a deficit in synaptic vesicle fusion followed by that in quantal size and vesicle docking/priming, revealing specific functional deteriorations in synaptic transmission. Pharmacological stimulation of synaptic transmission can improve motor activity in aged animals. These results uncover a critical role for the nervous system in age-dependent motor activity decline in C. elegans and provide insights into how functional aging occurs in this organism.
•As animals age, they gradually lose motor activity, but the mechanism is unclear•In C. elegans, the nervous system, but not muscles, functionally declines in early life•Motor neurons develop specific deficits in synaptic transmission in early life•Chemical stimulation of synaptic transmission improves motor activity in old worms
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